This invention relates to reagents and methods for rapidly and quantitatively assaying the concentration of analytes in biological samples. More particularly, this invention includes stabilized vancomycin bidentate conjugates and other glycopeptide antibiotic bidentate conjugates and uses thereof in immunoassay formats for assaying the concentration of vancomycin in a test sample.
The ability to determine the concentration of therapeutic agents in a biological sample is of broad importance in medicine. For example, glycopeptide antibiotics such as vancomycin, eremomycin, ristocetin A, etc., are clinically important in the treatment of post-surgical staphylococcal infections. However, even these otherwise beneficial drugs can induce life-threatening symptoms if abused or mis-dosed. Indeed, the adverse side-effects of these antibiotics, such as nephrotoxicity and ototoxicity, have been well-documented (Costa Silva, V. L. et al., Renal Physiol. 10:327-337 (1987); Fee, W. E. et al., Rev Infect. Dis. 5 (Suppl. 2):S304 (1983); Lane, A. Z. et al., Amer. J. Med. 62:911 (1977)). Thus, a narrow margin exists between the therapeutic dosage and toxicity-inducing overdosages (Witchitz, J. L. et al., Nour. Presse Med. 11:489-491 (1982); Damien, J. M. et al., Ann. Biol. Clin. 48:217-220 (1984). In view of the wide use of these therapeutic agents, and the importance of accurately assaying the concentration of antibiotics in patient samples, a variety of methods have been developed to permit the screening of large numbers of patients.
Immunoassays are assay systems that exploit the ability of an antibody to specifically recognize and bind to a particular analyte or xe2x80x9cantigen.xe2x80x9d An antigen is a substance which is capable of inducing an immune response, i.e., antibody production, when introduced into an animal or human body. The region of an antigen that is recognized by an antibody and to which the antibody binds is referred to as an xe2x80x9cepitope.xe2x80x9dAlthough large molecules such as proteins or other xe2x80x9cantigensxe2x80x9d possess multiple epitopes, low molecular weight molecules such as most pharmacological agents possess only a single epitope. Such low molecular weight molecules are referred to herein as xe2x80x9chaptens.xe2x80x9d
The simplest immunoassay involves merely incubating an antibody that is capable of binding to a predetermined molecule (i.e., the xe2x80x9canalytexe2x80x9d) with a sample that is suspected to contain the analyte. The presence of the target molecule is determined by the presence, and is proportional to the concentration, of any immune complexes that form through the binding of antibody and the analyte. In order to facilitate the separation of such immune complexes from the unbound antibody initially present, a solid phase is typically employed. For example, in particle enhanced immunoassays, either the antibody or the antigen is immobilized on latex particles. The presence of the target molecule is then determined by incubating the immobilized antibody or antigen in the presence of the analyte-containing sample.
Target molecules that have become bound to the immobilized antibody can be detected in any of a variety of ways. For example, the support can be incubated in the presence of a labeled, second antibody (i.e., a xe2x80x9csandwichxe2x80x9d immunoassay) that is capable of binding to a second epitope of the target molecule. Immobilization of the labeled antibody on the support thus requires the presence of the target, and is proportional to the concentration of the targets in the sample. In an alternative assay, the sample is incubated with a known amount of labeled targets and antibody binding sites. The presence of any target molecules in the sample competes with the labeled target molecules for the antibody binding sites. Thus, the amount of labeled target molecules that are able to bind the antibody is inversely proportional to the concentration of target molecules in the sample. This is known as a competitive immunoassay.
The various immunoassay formats can be further divided into two main classes depending upon whether the assay requires the separation of bound species from unbound species. Heterogeneous immunoassays require such purification and, hence, entail a separation or isolation step. In contrast, homogeneous assays are designed such that the removal of bound species from unbound species is unnecessary. Because homogeneous assays lack a separation step, and are more easily automated, they are more desirable than heterogeneous assays in applications that entail the screening of large numbers of patients.
If the immune complex is large enough, it will become capable of scattering light, or of spontaneously precipitating. In such cases, agglutination, nephelometric, or turbidimetric immunoassay methods may be employed. Nephelometric methods measure the light scattered by a suspension of particles or reflected toward a detector that is not in the direct path of light (Sternberg, J. C., Clin. Chem. 23:1456-1464 (1977)). In contrast, turbidimetric methods measure the reduction of light transmitted through the suspension of particles or aggregates. The reduction is caused by reflection, scatter, and absorption of the light by the aggregates. In both nephelometry and turbidimetry, the rate of change in light scatter may also be measured, and provides an indication of the amount of antigen present. Agglutination assays measure the precipitation of antibody-antigen complexes. Such assays can be extremely sensitive and are amenable to automation. Because nephelometric and turbidimetric methods do not require the separation of the initially present antibody from the immune complexes formed in the assay, such assays are homogenous immunoassays.
The requirement of producing large immune complexes has limited the applicability of nephelometric, turbidometric, or agglutination immunoassays to high molecular weight molecules, such as proteins, that possess several epitopes (i.e. antibody binding sites). In particular, many haptens such as therapeutic agents have only a single epitope and, as such, are incapable of forming the large immune complexes needed for such immunoassays.
Two approaches have been exploited to define agglutination assays for haptens. One approach is a particle enhanced immunoassay involving the agglutination of antibody-coated particles with a polyepitopic species or a developer antigen containing at least two covalently coupled hapten analogs (e.g., a protein carrier, such as BSA) (Mongkolsirichaikul, D. et al., J. Immunol. Meth. 157:189-195 (1993)). The agglutination reaction requires the use of a developer antigen or a polyepitopic species because a molecule that has only one epitopic site cannot bind two antibodies, and hence cannot cross-link two antibodies together. Such cross-linking is, however, an essential step in the formation of large immune complexes. The second particle enhanced approach involves the agglutination of hapten-coated particles and antibody for the agglutination reaction.
With either method, the hapten or drug in the sample competitively binds to the antibody binding sites and results in inhibition or reduction of the immunoagglutination. Particle agglutination assays for therapeutic drugs and drugs of abuse which use hapten-coated particles are commercially available. Examples of such assays are PETINIA (Du Pont) and AbuScreen (Roche), Advisor (Abbott) and that of Mitsubishi.
A third solution to this problem has recently been described by Yan, et al. in U.S. Pat. No. 5,747,352, which is incorporated herein by reference. Yan et al. disclose a particle-enhanced homogeneous assay for aminoglycoside antibiotics, including vancomycin. The method is based on a latex-avidin bidentate assay for vancomycin using a biotinylated vancomycin bidentate conjugated to an avidin-latex particle. In the bidentate immunoassay method described by Yan et al., the biotinylated vancomycin/avidin latex particle conjugate is incubated with an anti-vancomycin antibody and a test sample. The inhibition of agglutination between the conjugate and the antibody indicates the presence of vancomycin in the sample.
The development of an assay kit for vancomycin, however, has met with great difficulty since vancomycin is known to be chemically unstable. For example, vancomycin forms several degradation products in aqueous solution at about pH 7 after several days. See, for example, FIG. 1, which provides an HPLC plot of a vancomycin solution at pH 7 after standing several days, showing the presence of the degradation products CDP1-M (crystalline degradation product, major form) and CDP1-m (crystalline degradation product, minor form). Thus, vancomycin in assay kit calibrators breaks down into CDP products, resulting in the loss of potency of the calibrator. Since calibrators are used for the purpose of obtaining a calibration curve, the use of calibrators containing partially degraded vancomycin will result in incorrect assay results. Further, vancomycin will dimerize by hydrogen bonding upon standing in solution. As a result, vancomycin calibrator kits are usually shipped in lyophilized form as with the EMIT(copyright) assay, or in a frozen state as with those from Abbott for the TDx(copyright) System.
Intermolecular dimerization of vancomycin molecules presents another problem in particle-enhanced immunoassays which employ vancomycin-immobilized particles, since vancomycin dimerization leads to agglutination of the vancomycin-immobilized particles prior to their use in the assay.
Nxcex1,Nxcex2-diacetyl-L-lysine-D-alanine-D-alanine (DALAA) is a tripeptide that is known to bind to free vancomycin. Other dipeptides and tripeptides such as acetyl-D-alanine-D-alanine (ADADA) and acetyl-D-leucine-D-alanine (ADLDA) are known to bind vancomycin, but with lower affinity constants. It is also known that dipeptide- or tripeptide-complexed vancomycin can dimerize in solution as well, forming, in the case of tripeptides, tripeptide-vancomycinxe2x80x94vancomycin-tripeptide complexes (Mackay, et al., J. Am. Chem. Soc., (1994) 116: 4581-4590; Mackay et al., J. Am. Chem. Soc., (1994) 116: 4573-4580; Gerhard, et al., J. Am. Chem. Soc., (1993) 115: 232-237; Zheng Shi, et al., J. Am. Chem. Soc., (1993) 115: 6482-6486; J. Am. Chem. Soc., Popieniek, et al., (1991) 113: 2264-2270; Groves, et al., Structure, (1994) vol. 2, No. 8, 747-754; Waltho, et al., J. Am. Chem. Soc., (1989) 111: 2475-2480; Kannan, et al., J. Am. Chem Soc., (1988) 110: 2946-2953; Williams, et al., Biochemical Pharmacology, (1988) vol. 37, No. 1, 133-141; Nieto et al., Biochem. J., (1971) 123:789-803). Mackay et al. (J. Am. Chem. Soc. (1994) 116: 4581-4590) have reported that while the dipeptide or tripeptide such as DALAA prevents or reduces vancomycin from forming CDP1 products, it also enhances the dimerization of vancomycin molecules, forming, in the case of dipeptides, dipeptide-vancomycinxe2x80x94vancomycin-dipeptide complexes.
There still exists a need for stabilized solutions of vancomycin-immobilized particles for assaying vancomycin in test samples. This need is met by the present invention, which provides stable vancomycin-immobilized particles, formulations comprising the stabilized particles and improved immunoassays employing such formulations.
This invention relates to novel stabilized formulations comprising glycopeptide antibiotics such as vancomycin immobilized on carrier particles and, more particularly, to methods for stabilizing glycopeptide antibiotic bidentate conjugate formulations for use in assaying glycopeptide antibiotics in a test sample. The invention also relates to assay formats that utilize such stabilized formulations. The glycopeptide antibiotic bidentate conjugates of this invention comprise glycopeptide antibiotics including, but not limited to, vancomycin, eremomycin, ristocetin A, and other glycopeptide antibiotics having structures similar to vancomycin.
Accordingly, one aspect of this invention provides methods for preparing stabilized vancomycin conjugate formulations, the method comprising:
(a) forming a vancomycin conjugate comprising a vancomycin member bound to a ligand, the ligand being bound to a ligand-binding partner immobilized on a solid support, wherein the conjugate is formed under conditions that eliminate intermolecular hydrogen bonding between the vancomycin members;
(b) mixing the conjugate with at least one stabilizing agent that prevents dimerization between vancomycin members, wherein the pH of the mixture is between about pH 7 to pH 9;
(c) heating the mixture at a temperature between about 40xc2x0 and 50xc2x0 C. for about 3 to 14 days to ensure colloidal stability; and
(d) storing the heat-treated mixture obtained in step (c) in a diluent buffer having a pH of about 6.5-8.5.
Another aspect of the present invention provides stabilized vancomycin conjugate formulations prepared according to the method of this invention, comprising:
a) a bidentate conjugate comprising a vancomycin member bound to a ligand, the ligand being bound to a ligand-binding partner immobilized on a solid support; and
b) at least one stabilizing agent that prevents dimerization between vancomycin members, wherein the bidentate conjugate and the stabilizing agent are dissolved in a diluent buffer having a neutral pH.
The stabilized vancomycin conjugate formulations of the present invention may be used in particle enhanced immunoassays for the detection of vancomycin in a sample. The stabilized vancomycin conjugate formulations are particularly suited for competitive immunoassays. Accordingly, another aspect of the present invention provides an immunoassay for the detection of vancomycin in a test sample, comprising:
(a) providing a stabilized vancomycin conjugate formulation comprising:
(i) a bidentate conjugate comprising a vancomycin member bound to a ligand, the ligand being bound to a ligand-binding partner immobilized on a solid support; and
(ii) at least one stabilizing agent that prevents dimerization between vancomycin members, wherein the bidentate conjugate and the stabilizing agent are dissolved in a conjugate diluent having a neutral pH;
(b) providing an antibody that is immunoreactive with vancomycin;
(c) mixing the sample with the vancomycin conjugate formulation and the antibody to form a reaction mixture;
(d) incubating the reaction mixture under conditions that allow binding of the antibody to the vancomycin contained in the sample or the vancomycin member of the bidentate conjugate; and
(e) determining the amount of the vancomycin member of the bidentate conjugate that bound with the antibody, wherein the amount is inversely proportional to the concentration of the vancomycin in the sample.
The present invention further provides a test kit for use in an immunoassay for determining the amount of vancomycin in a test sample, wherein one of the components of the test kit is a stabilized vancomycin conjugate formulation of this invention.
The invention further provides a novel rate enhancer for enhancing binding between an analyte in a test sample and an anti-analyte antibody in an immunoassay. The rate enhancer is particularly useful in particle-enhanced immunoassays.
The invention further provides a novel dose-response modulators for use in immunoassays. The dose-response modulators enable modulation of immunoassay sensitivity so as to obtain an assay that exhibits a more desirable reaction rate and dose response.
Additional objects, advantages and novel features of this invention shall be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following specification or may be learned by the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities, combinations, and methods particularly pointed out in the appended claims.